The present invention relates to polysaccharide-degrading enzymes, especially to the enzymes, in particular, to a lichenase enzyme which is capable of degrading (1,3-1,4)-.beta.-glucans and sequences encoding lichenase enzymes.
Hemicellulose (non-cellulosic polysaccharides including glucans, mannans and xylan) is the major constituents of plant cell walls. The mixed-linked 1,3-1,4-.beta.-glucans form the major part of cell walls of cereals like oat and barley. .beta.-Glucans consist of glucose units jointed by .beta.-1,4 and .beta.-1,3 linkages, and include lichenan and barley .beta.-glucan. .beta.-Glucan accounts for up to 70% of the cell wall in barley endosperm (Guliga and Brant, 1986).
Endo-1,3-1,4-.beta.-D-glucanohydrolase (1,3-1,4-.beta.-glucanase, lichenase) cleaves .beta.-1,4 linkages adjacent to .beta.-1,3 in glucans yielding chiefly cellobiosyltriose and cellotriosyltetraose (Fleming and Kawami, 1977; Anderson and Stone, 1975). .beta.-Glucanase is especially interesting to the brewing industry because .beta.-glucans cause problems in filtration processes (Godfrey, 1983). .beta.-glucanase also has application in the poultry industry; it has been added to broiler chick feedstuffs to improve digestibility (White et al., 1983). .beta.-Glucanases have been cloned from several Bacillus species, including Bacillus subtilis (Murphy et al., 1984), B. amyloliquefaciens (Hofemeister et al., 1986), B. macerans (Borriss et al 1990), B. licheniformis (Lloberas et al., 1991), B. brevis (Louw et al., 1991), B. polymyxa (Gosalbes et al., 1991), and from other genera, including Clostridium thermocellum (Schimming et al., 1992; Zverlov et al., 1992), Fibrobacter succinogenes (Teather and Erfle, 1990), Ruminococcus flavefaciens (Flint et al., 1993), Rhizobium meliloti (Berker et al., 1993, and Cellvibrio mixtus (Sakellaris et al., 1993). A cDNA clone encoding barley 1-glucanase has been isolated and sequenced from germinating barley (Fincher et al., 1986).
Unlike endo-1,4-.beta.-D-cellulases which are widely distributed in various organisms, 1,3-1,4-.beta.-D-glucanases are known to be produced only by plants and certain bacteria (Borriss et al., 1990; Fincher et al., 1986). No fungal 1,3-1,4-.beta.-glucanases which lack the ability to degrade 1,3-(1,4)-glucans are believed to have been discovered prior to the present invention.
Obligately anaerobic fungi are part of the natural microflora of the alimentary tract of many herbivorous mammals (Orpin and Joblin, 1988). Since the first strictly anaerobic and filamentous fungus Neocallimastix frontallis was isolated in 1975 from the rumen of a sheep (Orpin, 1975), at least thirteen different anaerobic fungi have been isolated from ruminant and nonruminant herbivores (Chen et al., 1995a). Anaerobic fungi are divided into two groups based on morphology. One is monocentric, and it includes Neocallimastix (Orpin, 1975), Caecomyces (Wubah and Fuller, 1991), and Piromyces species (Barr et al., (1989) Can. J Botany 67:2815-2824); the other is polycentric and it contains Orpinomyces (Barr et al., (1989) supra), Anaeromyces (Breton et al., 1990), and Ruminomyces (Ho and Bauchop, 1990). The anaerobic fungi produce a variety of enzymes that degrade plant materials ingested by the host animals (Borneman et al., 1989). The physical association with the lignocellulosic tissues of plant fragments, and the ability to penetrate and weaken the plant tissue in vivo (Akin et al., 1983) suggest that the fungi are involved in degradation of digesture and that they play an important role in the rumen ecosystem. Several cellulases and xylanases have been cloned and sequenced from both monocentric Neocallimastix patriciarum (Gilbert et al., 1992; Zhou et al., 1994; Black et al., (1994) Biochem. J 299:381-387; Denman et al., (1996) Appl. Envir. Microbiol 62:1889-1896; Piromyces sp. (Fanutti et al., 1995) and polycentric Orpinomyces PC-2. A mannanase was cloned and sequenced from Piromyces sp. (Fanutti et al., 1995).